CCR7 Receptor Expression in Mono-MAC-1 Cells: Modulation by Liver X Receptor α Activation and Prostaglandin E2

Cell migration via chemokine receptor CCR7 expression is an essential function of the immune system. We previously showed that prostaglandin E2 (PGE2), an important immunomodulatory molecule, increases CCR7 expression and function in monocytes. Here, we explore the role of the liver X receptor α (LXRα) activation on CCR7 expression in Mono-Mac-1 (MM-1) cells in the presence of PGE2. To do this, MM-1 cells were stimulated with the LXRα synthetic agonist T0901317 in the presence or absence of PGE2. CCR7 mRNA transcription was measured using quantitative RT-PCR and protein expression was examined using flow cytometry. CCR7 function was analyzed using migration assays in response to CCL19/CCL21, which are natural ligands for CCR7. Our results show that agonist-mediated activation of LXRα in the presence of PGE2 increases CCR7 mRNA transcription and MM-1 cell migratory capacity in response to CCL19/21. In addition, our results demonstrate that engagement of the E-prostanoids 2 and 4 (EP2/EP4) receptors present on MM-1 cells is responsible for the observed increase in CCR7 mRNA expression and function during LXRα activation. Examination of monocyte migration in response to lipid derivatives such as PGE2 and oxysterols that are produced at sites of chronic inflammation would contribute to understanding the excessive monocyte migration that characterizes atherosclerosis.


Introduction
Inflammatory monocytes are rapidly recruited to sites of inflammation, but their excessive and/or prolonged recruitment hinders the resolution of inflammation and is a hallmark of numerous diseases. Chemokines CCL19 and CCL21, which are important for cellular migration, are expressed by lymphatic endothelia as well as within lymph nodes by stromal cells, endothelial cells, and dendritic cells (DCs) [1][2][3][4]. These chemokines are the natural ligands of CCR7, which is expressed in DCs [5], T and B cells [6], and monocytes [7]. Mice deficient in CCL19, CCL21, or CCR7 demonstrate defective DC trafficking and altered immune responses [8,9]. PGE 2 modulates immune responses both in vitro and in vivo [10]. A marked increase in PGE 2 production (as high as 10 −4 M) is generated in response to a variety of immunological stimuli and infections with different pathogens (reviewed in [11]). The immunomodulatory molecule PGE 2 appears to have a dual role in DC migration by regulating CCR7 expression and activity. Maturation-induced upregulation of CCR7 surface expression is not sufficient for monocytederived DCs (MoDCs) to migrate toward CCL19 and CCL21 [12,13]. Indeed, MoDC migration toward CCL19 and CCL21 was readily observed upon maturation in the presence of the proinflammatory mediator PGE 2 . However, PGE 2 did not alter expression levels of CCR7 on mature DCs [12,13]. In human monocytes, PGE 2 affects CCR7 mRNA expression and function [6,14].
In macrophages as well as in DCs, oxysterol-mediated activation of the nuclear liver X receptor (LXR) has been shown to modulate innate immunity and tumor growth (reviewed in [15]). LXR is expressed ubiquitously, whereas LXR is expressed in the liver, adipose tissue, adrenal glands, intestine, lungs, and cells of the myelomonocytic lineage [16].
Our results show that PGE 2 and synthetic LXR ligand, T0901317, strongly increase MM-1 cell migratory capacity in response to CCL19/21. Examination of monocyte migration in response to lipid derivatives, produced during chronic inflammation, would contribute to understanding the excessive monocyte migration that characterizes atherosclerosis.

Blood Monocyte Isolation.
Total blood mononuclear cells were isolated from the blood of healthy donors using lymphocyte separation Medium 1077 (Sigma) and washed twice in Hank's balanced salt solution (Wisent). Cells were cultured in RPMI 1640 medium, 20% heat-inactivated FBS, and 10% heat-inactivated human serum for 2 h before use. Monocytes were washed from nonadherent cells with phosphatebuffered saline (PBS). Monocytes were then enriched from peripheral blood mononuclear cells using the MACS Monocyte Isolation Kit II and MACS LS Columns (Miltenyi Biotec, Auburn, CA, USA), yielding an average purity of 98%. The purity was assessed by flow cytometric analyses as recommend by the manufacturer, and isolated monocytes were fluorescently stained with CD14-FITC and anti-Biotin-PE that labeled nonmonocytes. Blood monocytes were stimulated for the indicated times using 1 M PGE 2 and/or 1 M T0901317.  (Table 1), 0.1 mM dNTPs, 2 mM or 3.5 mM MgCl 2 , and 1.25 units of Omni Klentaq (Enzymatics, Beverly, MA, USA). In each reaction, we used 8 L of a 1 : 2 dilution of each cDNA (dilutions were performed using molecular grade sterile water (Wisent)). PCR cycling conditions consisted of an initial denaturation at 95 ∘ C for 3 minutes and 40 cycles of 95 ∘ C for 10 sec, 60 ∘ C for 40 sec, 72 ∘ C for 40 sec, and a melting curve at 95 ∘ C for 10 sec, 65 ∘ C for 5 sec, and 95 ∘ C for 5 sec.

Chemotaxis
Assays. MM-1 cell chemotaxis was measured by migration through a polycarbonate filter (5 m pore size) in a 96-well transwell chambers (Millipore, Nepean, ON, Canada). The lower chamber contained either 150 L of a 300 ng/mL dilution of chemokine CCL19/CCL21 in RPMI 1640 media without FBS, NEAA, and sodium pyruvate, but complemented with 0.25% BSA, or media alone as a spontaneous migration control. The upper chamber contained 2.5 × 10 5 cells in 75 L of medium. Chambers containing MM-1 cells were incubated for 4 h at 37 ∘ C. An aliquot (150 L) of cells that migrated to the bottom chamber was mixed with 1x PBS (150 L) and counted using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) by acquiring events for a fixed period of 60 seconds using CellQuest Software (BD Biosciences). The percentage of migrated cells was calculated as follows: the number of migrated cells in response to media only was subtracted from the number of migrated cells in response to CCL19/CCL21. This number was normalized to the total input of cells. Each experiment was performed in triplicate and repeated at least three times.
2.6. Flow Cytometry Analyses. MM-1 cells were collected and washed twice with PBS supplemented with 3% bovine serum albumin (BSA). Fc receptors were blocked for 15 minutes at room temperature using 100 L of human serum (diluted 1 : 5 in 1x PBS) for 1 × 10 6 cells. Cells were then washed twice in PBS plus 3% BSA. Cells were labeled with an anti-CCR7 antibody conjugated with allophycocyanin (APC), or corresponding isotypes as a negative control, for 45 minutes on ice in the dark. Cells were then washed twice with PBS plus 3% BSA and centrifuged 5 minutes at 10 g at 4 ∘ C. For intracellular experiments, cells were collected and washed with buffer (1x PBS plus 1% BSA and 0.02% sodium azide). Fc receptors were blocked using 100 L human serum for 15 minutes at room temperature and washed with wash buffer. Cells were fixed with 200 L 4% paraformaldehyde for 15 minutes at 4 ∘ C and again washed with wash buffer. Cells were permeabilized by adding 200 L of 1% saponin to the wash buffer and then incubated for 45 minutes on ice with anti-CCR7 coupled with APC (Abcam, San Francisco, CA, USA) or the corresponding isotype controls. Cells were then washed twice with wash buffer. Fluorescence was read using a BD FACSCalibur flow cytometer (BD Biosciences) and results were analyzed using CellQuest software (BD Biosciences).

Statistical Analyses.
Each experiment was performed at least three times. Statistically significant differences between experimental groups were evaluated using paired -tests and < 0.05 was considered statistically significant. Computations were performed using GraphPad PRISM version 6.0 statistical software (GraphPad, San Diego, CA, USA).

PGE 2 and LXR Activation Upregulate CCR7 mRNA Production and Function without Affecting CCR7 Surface
Expression in MM-1 Cells. MM-1 is a human cell line with the properties of blood monocytes that can be used as a model system to study monocytic functions in vitro [20]. We first used real-time qPCR to examine whether PGE 2 and LXR activation could modulate CCR7 transcription in MM-1 cells. MM-1 cells were treated with 1 M PGE 2 and 1 M T0901317, a synthetic LXR agonist, for 8 or 24 h (Figure 1(a)). As previously observed, PGE 2 induces CCR7 mRNA expression with a maximal effect after stimulation for 8 h [7]. Treatment of MM-1 cells with T0901317 alone does not modify the expression of CCR7 mRNA. Interestingly, we found that cells treated with a combination of PGE 2 and T0901317 showed significantly upregulated CCR7 mRNA transcription.
Next, we determined whether MM-1 cells can autoregulate LXR expression as previously demonstrated in macrophages [21]. LXR transcripts were measured using real-time qPCR following stimulation with 1 M PGE 2 and 1 M T0901317 (Figure 1(b)). We observed an increase in LXR levels following treatment with PGE 2 or T0901317 alone. mRNA production plateaued after 8 h of stimulation with PGE 2 or T0901317 alone. However, MM-1 cells that were stimulated with a combination of PGE 2 and T0901317 showed increased levels of LXR mRNA after 24 h compared to cells stimulated with PGE 2 or T0901317 alone.
We also investigated whether PGE 2 in combination with T0901317 affects the expression of one LXR target gene, ABCG1 (Figure 1(c)). As expected, ABCG1 mRNA levels were augmented when cells were treated with T0901317 alone. PGE 2 had no effect on ABCG1 transcription. However, MM-1 cells treated with a combination of PGE 2 and T0901317 showed significantly reduced levels of ABCG1 transcription.
We next established whether increased mRNA production correlates with CCR7 receptor function in MM-1 cells. MM-1 cells were treated with the stimulants for 24 or 48 h before migration through polycarbonate filters (5 m pore size) for 4 h. The chemotaxis assay results showed that PGE 2 increases MM-1 cell migration to both CCR7 natural ligands CCL19 (Figure 2(a)) and CCL21 (Figure 2(b)) after treatment for 24 h. Although the migratory capacity of MM-1 cells is sustained in response to CCL21 after 48 h, migration in response to CCL19 appears transient. The LXR agonist alone did not affect the migratory capacity of MM-1 cells in response to CCL19 whereas 24 h treatment with T0901317 significantly increased migration in response to CCL21. In contrast, migration of MM-1 cells treated with PGE 2 and T0901317 for 48 h was increased compared to untreated cells or cells treated with PGE 2 alone. For all chemotaxis assays, we confirmed the specificity of migration by incubating PGE 2 -and T0901317-stimulated cells with a blocking antibody against human CCR7 for 10 min prior to migration assays. Blockade of CCR7 at the MM-1 cell surface completely abolished specific migration to CCL19 and CCL21 (data not shown). To further investigate the effects of PGE 2 and T0901317 on CCR7 expression, we repeated migration assays with freshly isolated human blood monocytes. Results showed that monocyte migration toward CCL19 and CCL21 is increased following treatment of blood monocytes with PGE 2 and T0901317 (Figure 3).
We aimed at establishing whether the CCR7 receptor is expressed at the cell surface of PGE 2 -and T0901317stimulated MM-1 cells. Cells were incubated in the presence or absence of PGE 2 and T0901317 for 24 and 48 h. Surface expression of CCR7 was analyzed using flow cytometry. Our results showed that MM-1 cells basally express CCR7 (13.57% with a MFI (mean fluorescence intensity) of 4.94). However, after 24 h, CCR7 cell surface expression was upregulated by 1 M PGE 2 (31.66% with a MFI of 7.82) but not by T0901317 (13.11% with a MFI of 4.83) (Figure 4(a), upper panel). Similar results were observed after 48 h (Figure 4(b), lower panel). Statistics for CCR7 cell surface expression are presented in Table 2. Because CCR7 cell surface expression was not upregulated following the addition of PGE 2 and T0901317, we performed intracellular flow cytometry assays after 24 h (Figure 4(b), upper panel) and 48 h (Figure 4(b), lower panel)

The EP 2 and EP 4 Receptors Are Involved in LXR Activation and PGE 2 -Induced CCR7 Transcription and Functional
Migration. We previously showed that monocytes primarily express two PGE 2 receptors, EP 2 and EP 4 [7]. Moreover, we demonstrated that both receptors are implicated in PGE 2induced CCR7 upregulation in MM-1 cells [7]. Thus, using pharmacological agonists for PGE 2 receptors, we next determined whether EP 2 and/or EP 4 play a role in PGE 2 -and T0901317-induced CCR7 migration ( Figure 5). Treatment of MM-1 cells with the EP 2 and EP 4 agonist 11-deoxy-PGE 1 increased CCR7 mRNA levels compared to untreated cells ( Figure 5(a)). Although 17-PT-PGE 2 is described as   Total RNA was extracted and CCR7 transcripts were detected using real-time qPCR. Data represent mean ± SD of three independent experiments. Chemotaxis assays in response to 300 ng/mL CCL19 (b) or CCL21 (c) were performed using MM-1 cells stimulated for 48 h with 1 M T0901317. The mean number of spontaneously migrating cells (that migrated to media alone) was subtracted from the number of cells that migrated in response to CCL19. Data represent mean ± SD of three independent experiments. * < 0.05, * * < 0.01, * * * < 0.001, and * * * * < 0.0001. results showed that 17-PT-PGE 2 significantly enhanced CCR7 expression whereas butaprost alone or in combination with T0901317 slightly increased CCR7 RNA levels compared to control. To determine which PGE 2 receptors regulate the migratory response of T0901317-treated monocytes to CCL19 and CCL21, chemotaxis assays were performed on MM-1 cells in response to CCL19 (Figure 5(b)) or CCL21 (Figure 5(c)). Our results indicated that MM-1 cells migrated efficiently toward CCL19 or CCL21 when butaprost, 11-deoxy-PGE 1 , and 17-PT-PGE 2 were added to the milieu. However, the most remarkable migration occurred when PGE 2 -treated MM-1 cells were cultivated in the presence of T0901317.
Because EP 2 and EP 4 receptors activate adenylate cyclase (AC), which then increases intracellular cAMP levels [23], we next assessed whether forskolin, a pharmacological activator of AC, contributed to the observed induction of CCR7 mRNA (Figure 6(a)). MM-1 cells were stimulated with 100 M forskolin for 8 h or 24 h in the presence or absence of T0901317. We found that forskolin alone increases CCR7 mRNA transcription whereas forskolin combined with T0901317 did not affect CCR7 expression. AC activation did not modulate CCR7 mRNA production in T0901317-treated MM-1 cells. Thus, we searched for alternative signaling pathways. MM-1 cells were treated with H-89 (a PKA inhibitor), LY294002 (a PI3K inhibitor), or PD98059 (a MEK inhibitor) before the addition of PGE 2 and T0901317 for 48 h. Cells were used in migration assays in response to CCL19 or CCL21 (Figure 6(b)). The results showed that selective inhibition of PKA and MEK did not affect CCR7-dependent migration of MM-1 cells in response to CCL19 or 21. In contrast, inhibition of PI3K further enhanced MM-1 cell migration mediated by PGE 2 and T0901317.

Discussion
In addition to modulating cholesterol homeostasis, LXRs have emerged as important regulators of inflammatory gene expression and innate immunity [21]. In inflammation, PGE 2 is of particular interest because its deregulation is associated with the pathogenesis of various diseases and numerous tumor types [11,24]. In this study, we showed for the first time that PGE 2 in combination with LXR activation strongly 8 International Journal of Inflammation increased the CCR7-dependent migratory capacity of MM-1 cells, a monocytoid cell line. We showed that although CCR7 mRNA levels are upregulated following treatment with PGE 2 and T0901317, there was no change in CCR7 cell surface expression. In addition, our results indicate that EP 2 and EP 4 receptors are implicated in PGE 2 -mediated CCR7 transcription required for upregulating the migratory capacity of MM-1 cells in response to CCL19 and CCL21. In mature DCs, it has been previously shown that activation of LXRs interferes with CCR7 expression, resulting in a dampened antitumor immune response [18]. Recently, Bruckner et al. [19] demonstrated that PGE 2 rescues the migratory capacity of DCs cultivated in the presence of LXR ligands to migrate toward CCR7 ligands. In contrast to these results, LXR activation in MM-1 cells by the synthetic ligand T0901317 does not modify CCR7 expression (Figure 1(a)) or CCR7-dependent migration in response to CCL19 (Figure 2(a)) or CCL21 (Figure 2(b)). However, the combination of PGE 2 and T0901317 significantly increased CCR7 mRNA production and MM-1 cell migration compared to cells treated with PGE 2 or T0901317 alone. In addition, blood monocytes treated PGE 2 and T0901317 migrate in response to CCR7 specific ligands compared to untreated monocytes ( Figure 3). Importantly, MM-1 surface expression of CCR7 (Figure 4(a)) did not correlate with migration efficiency (Figure 2). Indeed, other studies have shown  that PGE 2 significantly enhances DC migration through an unknown mechanism that does not depend on the magnitude of CCR7 expression [12,25,26]. This phenomenon is also observed with MoDCs matured in the presence of both PGE 2 and T0901317 [19]. Thus, our results in monocytes are consistent with those observed by other groups in DCs. In addition, flow cytometry analyses were also performed to detect intracellular changes in CCR7 protein expression (Figure 4(b)). However, no changes in expression were detectable. Together, our results demonstrate that LXR activation and PGE 2 stimulation of MM-1 cells profoundly affect monocyte migration in response to CCL19 and CCL21. Further studies are required to examine the effect of LXR activation in the presence of PGE 2 on CCR7-dependent migration of freshly isolated blood monocytes from healthy donors.
In macrophages, LXR activation results in the synthesis of the cholesterol efflux transporter ABCG1 as well as of LXR itself through an autoregulatory mechanism [21]. In DCs, PGE 2 downregulated basal expression of LXR but also inhibited T0901317-mediated autoinduction of LXR [19]. In addition, T0901317-mediated ABCG1 induction was also significantly reduced in MoDCs matured in the presence of PGE 2 [19]. In our study, we observed that PGE 2 did not modulate ABCG1 mRNA production whereas T0901317 alone significantly affected transcription (Figure 1(b)). In contrast, in MM-1 cells stimulated with PGE 2 and T0901317 for 24 h, we observed significantly reduced ABCG1 mRNA production. In MM-1 cells, LXR activation upregulated LXR mRNA expression but PGE 2 had no effect. In contrast to results observed in DCs [19], 24 h treatment with PGE 2 and T0901317 strongly increased LXR mRNA transcription. Our results demonstrated that the addition of PGE 2 to T0901317-treated MM-1 cells reduced ABCG1 mRNA production. Because LXR expression was increased after MM-1 cells were treated with PGE 2 and T0901317 (Figure 1(c)), the effect on ABCG1 is likely LXR -independent. It has been shown that ABCG1 and ABCG4 act in concert with ABCA1 to maximize removal of excess cholesterol from cells [27]. Taken together, our results suggest that the presence of PGE 2 and oxysterols negatively impacts cholesterol efflux in monocytes. Moreover, the presence of these two lipids derivatives may favor intracellular cholesterol accumulation, thereby leading to deregulation of cholesterol homeostasis in monocytes. However, further studies are needed to clarify the mechanisms underlying the observed modulation of ABCG1 by PGE 2 and LXR ligands in monocytes.
The EP 2 and EP 4 receptors were previously shown to be important for CCR7 expression in mature MoDCs [13,19,25] and monocytes [7]. Here, our results showed that PGE 2 binding to the EP 4 receptor subtype, and to a lesser extent EP 2 , triggered signals that led to CCR7 mRNA expression and CCR7-dependent migration of MM-1 cells cultivated in the presence of the LXR synthetic ligand ( Figure 5). Interestingly, our data showed that PGE 2 -and T0901317-upregulation of CCR7 expression and function is independent of the cAMP/PKA or MEK branches of EP 2 /EP 4 signaling ( Figure 6). However, we showed that inhibition of the PI3K pathway is responsible for enhanced CCR7dependent MM-1 cell migration. Thus, our results suggest the contribution of other signaling pathways.
In summary, our results demonstrate that PGE 2 , in combination with LXR activation, increased CCR7-dependent migration of MM-1 cells (Figure 7). Lipid derivatives, including PGE 2 and oxysterols, may also favor cholesterol accumulation in monocytes. Therefore, our results may have important implications regarding the mechanisms that contribute to atherosclerosis. However, further studies are needed to better understand the role of lipid derivatives produced during inflammation.